1. Field
The present disclosure relates to a method and device for regulating fluid flow in microfluidic devices. In particular, it relates to methods for providing cells to a microfluidic device wherein fluid regulation relies on the equalization of pressures and a microfluidic device for performing the same.
2. Description of Related Art
Automated systems to perform fluorescence experiments on cells have been around for a while. Molecular Devices' FLIPR and FlexStation systems, for instance, can perform hundreds of cellular calcium assays simultaneously. These, however, do not have single cell sensitivity. Assays on thousands of single cells can be performed, but with the same conditions stimulating all of them (Teruel and Meyer. 2002, Science, 295, 1910-1912). Conditions can also be varied in experiments, but this is currently possible only with either low throughput (Wheeler, et al., 2003, Analytical Chemistry, 75, 3581-3586) or with a solute gradient (Chung et al., 2005, Lab on a Chip, 5, 401-406). PDMS (poly-dimethylsiloxane) microfluidic devices have enabled inexpensive rapid prototyping of sophisticated microfluidic applications (Unger, et al., 200, Science, 288:113-116; Thorsen, et al., 2002, Science, 298:58-584). Several disclosures have been made relating to microfluidic devices, their fabrication and uses thereof (U.S. Pat. No. 6,793,753; U.S. Pat. No. 6,899,137; U.S. Pat. No. 6,929,030, U.S. Pat. No. 7,040,338). U.S. Pat. No. 7,040,338 is incorporated herein by reference in its entirety.
Observing cells singly rather than as an ensemble is important since cells often have digital, stochastic responses to external stimuli (Elowitz et al., 2002, Science, 297, 1183-1186; Lahav et al., 2004, Nature Genetics, 36, 147-150). The nature of these responses can be masked when populations of cells are observed as a whole. The ability to do different experiments on identically prepared cells is also important, since many variables that influence cellular responses are difficult to control from batch to batch, experiment to experiment.
The approach to the problem as described herein, is to seed cells into molded poly(dimethysiloxane) (PDMS) microchannels, and then manipulate the environment that the cells experience by switching the solutions that flow over them with pressure-actuated valves (Studert et al., 2004, J. of Applied Physics, 95, 393-398). In this way, several microchannels can be packed into one field of view on a microscope, so that several different experiments can be performed on cells which originate from a single batch and which have experienced essentially identical manipulations.
In order to make cellular experiments compatible with a valve-gated microfluidic network, the method and device disclosed herein provides a new way of seeding microfluidic devices with cells. Until now, all devices that need to use cells load them either one at a time from a dilute cell suspension (Wheeler, et al., 2003, Analytical Chemistry, 75, 3581-3586; Lee et al., 2005, Applied Physics Letters, 86, 223902), or in one pass with a concentrated pass (Andersson and van den Berg, 2003, Sensors and Actuators B, 92, 315-325; Taylor et al., 2005, Nature Methods, 2, 599-605). The former approach becomes impractical in situations where high throughput and large numbers of cells are required. The latter, on the other hand, can cause problems when microfluidic valves are used since some cells will inevitable be trapped in, and crushed by, those valves.
This problem with trapped or crushed cells is especially troublesome in experiments involving macrophages. Assays on macrophage cell lines are of particular interest, since they have rather large and complex signal transduction systems. Macrophages, however, are programmed to respond to signs of nearby cell death. Thus, in a microfluidic device, care must be taken to prevent killing cells upstream of the cells that are being experimented on. In addition, macrophages are inherently adherent, and there is a significant possibility of adhesion to most surfaces when they suspension is stagnant, so it is difficult to selectively remove macrophages after they have adhered to a surface.
The disclosure presented herein provides for a new method and device relating to a PDMS microfluidic device.